American Thoracic Society Documents

An Official American Thoracic Society Workshop

Report: Features and Measurements of Experimental
Acute Lung Injury in Animals
Gustavo Matute-Bello, Gregory Downey, Bethany B. Moore, Steve D. Groshong, Michael A. Matthay,
Arthur S. Slutsky, and Wolfgang M. Kuebler, on behalf of the Acute Lung Injury in Animals Study Group
THIS OFFICIAL WORKSHOP
SEPTEMBER 2010

REPORT OF THE

AMERICAN THORACIC SOCIETY (ATS)

CONTENTS
I. Statement of the Problem
II. What Is Being Modeled?
III. Methodology
IV. Results: Features and Measurements of ALI in Animals
V. Practical Aspects of Measuring Ali in Animals
VI. Critical Assessment of Selected Common Models of Lung
Injury
VII. Limitations
VIII. Summary and Conclusions
Acute lung injury (ALI) is well defined in humans, but there is no
agreement as to the main features of acute lung injury in animal
models. A Committee was organized to determine the main features
that characterize ALI in animal models and to identify the most
relevant methods to assess these features. We used a Delphi approach in which a series of questionnaires were distributed to a panel
of experts in experimental lung injury. The Committee concluded
that the main features of experimental ALI include histological
evidence of tissue injury, alteration of the alveolar capillary barrier,
presence of an inflammatory response, and evidence of physiological dysfunction; they recommended that, to determine if ALI has
occurred, at least three of these four main features of ALI should be
present. The Committee also identified key very relevant and
somewhat relevant measurements for each of the main features of
ALI and recommended the use of least one very relevant measurement and preferably one or two additional separate measurements
to determine if a main feature of ALI is present. Finally, the
Committee emphasized that not all of the measurements listed
can or should be performed in every study, and that measurements
not included in the list are by no means irrelevant. Our list of
features and measurements of ALI is intended as a guide for investigators, and ultimately investigators should choose the particular measurements that best suit the experimental questions being
addressed as well as take into consideration any unique aspects of
the experimental design.
Keywords: acute lung injury; animal model; disease models

I. STATEMENT OF THE PROBLEM

What is acute lung injury (ALI) in an animal? One would think
that the answer to this question is well established, considering
that a PubMed search in mid-2008 retrieved more than 14,000
entries for the query Lung Injury AND Animal, and that

Am J Respir Cell Mol Biol Vol 44. pp 725738, 2011

DOI: 10.1165/rcmb.2009-0210ST
Internet address: www.atsjournals.org

WAS APPROVED BY THE

ATS BOARD

OF

DIRECTORS

ON

considerable resources have been invested in the study of

experimental ALI during past decades. However, a closer look
at the published literature reveals that there is no universal
agreement as to the precise definition of ALI in experimental
animal models. Is an increase in the concentration of lung cytokines sufficient to indicate lung injury, or should there also be
evidence of cellular inflammation? If so, does it suffice to have
inflammatory cells in the interstitium, or should they also be
present in the airspaces? Or perhaps lung injury should, instead,
be determined based on changes in alveolar epithelial permeability? There is no consensus in the scientific community as to
what exactly constitutes ALI in an animal, in part because there
is no single marker or parameter that has sufficient sensitivity
and specificity to identify the occurrence of all forms and
severities of ALI. As a result, it is difficult for investigators to
determine if they have achieved (or prevented) lung injury in an
experimental model. For example, if an experimental drug
decreases neutrophil migration into the lungs but has no effect
on permeability changes, does it prevent lung injury or not? In
addition, a comparison of studies is difficult because of the
diversity of assays used by investigators to assess lung injury.
The same experimental challenge may or may not result in lung
injury, depending on how lung injury is defined by the investigators. Clearly, answers to such questions are relevant in an
area of research that is as important to patient care as ALI.
In humans, the definition of ALI is based on the following
well-defined set of clinical parameters developed by the American European Consensus Conference: acute onset, radiological
evidence of diffuse bilateral pulmonary infiltrates, a ratio of the
partial pressure of arterial oxygen to the fraction of inspired
oxygen (PaO2/FIO2) of less than 300, and no clinical evidence for
elevated pulmonary arterial pressure (1). However, these
criteria cannot be directly translated to experimental animals.
Although arterial blood gases, chest radiographs, cardiac echocardiography, and even catheterization can be performed in
small experimental animals, the equipment required for these
measurements is available only in a few laboratories. Furthermore, such measurements may be incompatible with the design
of many experimental systems. Thus, it is not practical to use
the American European Consensus Conference criteria in
animal studies, particularly in small animals. An alternative
approach would be to define ALI in animals based on histopathological criteria similar to those seen in humans with ALI.
In humans, the pathological correlate of ALI is diffuse alveolar
damage (DAD), characterized by inflammatory infiltrates,
thickened alveolar septae, and deposition of hyaline membranes
(2). However, as discussed below, none of the available animal
models of ALI reproduce all of the pathologic features of DAD

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in humans. Therefore, the criteria used to define ALI in humans

cannot be directly translated to most animal models of ALI.
The goal of the present workshop was to determine the main
features that characterize ALI in animals and then to identify
the most relevant measurements to assess these features. We
used a Delphi approach in which a series of questionnaires were
distributed among a panel of experts in experimental lung
injury. In this report, we discuss the main features of human
ALI that are being modeled in animals, describe the methodology used to identify the defining features of lung injury in
animals, discuss common methods of measuring lung injury,
critically assess standard models of lung injury, critically assess
standard models of lung injury, and finally, discuss the limitations of this approach.

II. WHAT IS BEING MODELED?

Ideally, an animal model of ALI should capture one or more
features of human ALI, including rapid onset (hours) after an
inciting stimulus, evidence of pulmonary physiological dysfunction (e.g., abnormalities of gas exchange, decreased lung compliance), histological evidence of injury to the lung parenchyma
(endothelium, interstitium, epithelium), and evidence of increased permeability of the alveolocapillary membrane. Any
one animal model is unlikely to encompass all of the salient
features of ALI/ARDS observed in humans. Thus, a key question
is how many of the features of human ALI need to be present in
an animal model for it to be classified as demonstrating ALI?
This is a complex issue without a right or wrong answer and is
dependent on many factors, including the specific experimental
question being addressed. To understand these issues, it is
important to review certain differences between animals and
humans that are pertinent to ALI and then to compare humans
and animals in terms of these variables.
Differences between Animals and Humans Relevant to ALI

There are many anatomical and physiological differences

between animals and humans that influence the response of
the lung to an acute injurious stimulus and affect the evaluation
of lung injury (3). Some of these are obvious; for example, the
respiratory rate of mice (250300 bpm) far exceeds that of adult
humans (1216 bpm), rendering absolute respiratory rate inadequate as a parameter of ALI in mice (4, 5). There are
important differences in the gross and microscopic anatomy
between rodent and human lungs. Mice have no bronchial
arteries. Both the alveoli and thickness of the bloodgas barrier
are smaller in the lungs of mice and rats compared with humans.
Mice and rat lungs have a lobar anatomy distinct from that of
humans and their lungs have fewer branches of the conducting
airways proximal to the terminal bronchioles. At the microscopic level, murine lungs differ from human lungs in that they
have a larger number of Clara cells in the distal airways;
extensive bronchial-associated lymphoid tissue, and a virtual
absence of submucosal glands beyond the proximal trachea (6).
In the setting of ALI, murine lungs rarely demonstrate typical
hyaline membranes in situations where there is clearly enhanced permeability of the alveolocapillary membrane. There
are also important differences in key elements of the inflammatory response, both cellular and humoral, between rodents
and humans. For example, mice have fewer circulating neutrophils (1025%) than humans (5070%) and do not express
defensins (6). The neutrophil repertoire of CXC chemokines
differs between rodents and humans (keratinocyte-derived
chemokine KC [CXCL1] and macrophage inflammatory protein
[MIP-2] in mice, cytokine induced neutrophil chemoattractant
[CINC] in rats, and IL-8 in humans) (7). In addition, critically ill

humans develop ALI/ARDS in the setting of a number of

interventions such as prolonged ventilation or hemodynamic
support, which are difficult to reproduce in animals. Finally,
animal studies frequently use young mice with no comorbidities,
whereas human patients tend to be older and have multiple
medical problems such as diabetes, coronary artery disease, renal
or hepatic insufficiency, and so forth. For all of these reasons, the
responses of animal and human lungs to an injurious stimulus
cannot be expected to be identical or perhaps even similar.
Comparison of Specific Parameters in the Assessment of ALI
in Animal Models

1. Assessment of the kinetics of lung injury. Given the relatively controlled nature of experimental models of ALI, the
time of exposure of the inciting stimulus (e.g., lipopolysaccharide, acid aspiration, hemorrhagic shock, or injurious mechanical ventilation) is usually known with precision. This differs
from the human situation where the time of exposure to an
inciting stimulus (e.g., bacterial infection with sepsis) is often
less clear. Nonetheless, in animal systems that seek to model
human ALI, maximal lung injury should be evident within 24
hours of exposure to the inciting stimulus to distinguish those
conditions that reflect more subacute or chronic lung injury.
2. Radiographic assessment of lung injury. The ability to
assess lung injury radiographically in animal models is constrained by several factors, including the small size of rodents
and the limited availability of radiographic facilities for animals
in most laboratories. Nonetheless, if available, radiographic
demonstration (plain X-ray, micro-computer tomography) of
bilateral and diffuse pulmonary infiltrates in animals represents
one parameter to assist in assessing the extent of lung injury.
This is more feasible for larger animals such as ferrets, rabbits,
dogs, or sheep.
3. Physiological assessment of lung injury. (i) Abnormalities of gas exchange. In humans, an elevated Aa gradient,
reflecting compromise of the gas exchange function of the lungs,
is one of the principal parameters used to diagnose, stratify, and
monitor patients with ALI or acute respiratory distress syndrome (ARDS). Although this is feasible and indeed routine in
larger animals, it is more difficult in rodents, and especially
mice, because of the difficulty of obtaining a sufficient sample
of arterialized blood without introducing other variables (e.g.,
trauma during sample collection or hypovolemia from removal
of a substantial fraction of the circulating blood volume). Noninvasive continuous monitoring of capillary or tissue oxygen
saturation using conventional microscopic or fluorescence enhanced oximetry or electron paramagnetic resonance spectroscopy is technically possible and is a useful parameter to monitor
if the appropriate instrumentation is available (8, 9).
(ii) Decreased lung compliance. Decreased lung compliance
due to pulmonary edema and atelectasis is a hallmark of human
ALI/ARDS and is an important and easily assessed parameter
in animal models. In mechanically ventilated animals, respiratory system compliance can be assessed in vivo during mechanical ventilation in a similar fashion to how it is measured in
humans or by determining the pressurevolume curve after
a recruitment maneuver. A particularly useful method is to use
commercially available ventilator systems that allow automated
measurement of respiratory mechanics and generate pressurevolume curves and measurement of compliance (10). These
systems can be programmed to automatically perform measurements at predetermined time intervals. These systems are
effective but remain expensive. Measurement of lung compliance in vivo requires an estimate of pleural pressure. An equally
acceptable method is to measure the pressurevolume curve of
lungs ex vivo.

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4. Assessment of increased permeability of the alveolocapillary membrane. Change in alveolocapillary membrane

permeability is one of the critical parameters that defines the
pathophysiology of ALI in humans and animals, but its measurement is fraught with technical pitfalls that vary based on the
method used. During the genesis of ALI/ARDS the selective
barrier function of the pulmonary endothelium and/or epithelium is lost due to injury or dysfunction. This is manifest by
leakage of protein-rich fluid from the vascular to the interstitial
and/or alveolar space. Many methods have been developed to
assess this alteration. The most commonly used methods include
(1) measurement of the concentration of albumin or other high
molecular weight proteins such as IgM in bronchoalveolar
lavage (BAL) fluid compared with that in plasma, (2) assessment of the leakage of an exogenous labeled high molecular
weight molecule (e.g., 125I-labeled albumin or fluorescent high
molecular weight dextran) or Evans blue dye (which binds to
albumin) into the alveolar space, and (3) the wet to dry weight
of the excised lungs. In larger animals, especially sheep, older
studies measured increased lymphatic flow and protein concentration in lymphatic fluid as an indicator of interstitial edema
(presumably an early phase of pulmonary edema). Each of
these is a valid method provided that appropriate corrections
for dilution of BAL fluid and/or intravascular blood volume are
made. Measurement of the total protein concentration of the
BAL fluid may reflect an increase in the permeability of the
alveolocapillary membrane, but it should be used with caution
as the sole measure of increased permeability, as further discussed in Section V.2 below.
5. Histological assessment of lung injury. One of the most
challenging aspects of using animal models of ALI pertains to
the histological assessment of lung injury. The pathological
hallmark of ALI in humans is diffuse alveolar damage (DAD)
(2, 11, 12). In humans, DAD is characterized by: an early exudative phase featuring neutrophil accumulation in the vascular,
interstitial, and alveolar spaces known as neutrophilic alveolitis); deposition of hyaline membranes composed of fibrin and
other proteinaceous debris as evidence that serum proteins have
entered and precipitated in the airspaces (i.e., disruption of the
alveolocapillary membrane); interstitial thickening; and the
formation of microthrombi (evidence of endothelial injury and
intraluminal activation of the coagulation cascade). This is followed by a proliferative phase characterized by alveolar epithelial cell hyperplasia and interstitial fibrosis. It is reasonable to
expect that animal models of ALI/ARDS should reflect some of
these features. However, no animal model completely reproduces
all of the histological features of DAD in humans (7, 13). The
presence of only a single histologic feature of ALI in an experimental animal (e.g., neutrophilic alveolitis) does not necessarily indicate a bona fide model of ALI and conversely, the
absence of one or more features does not exclude ALI. Despite
these differences, a semi-quantitative or quantitative assessment
of multiple histological features of ALI is critical for evaluating
the extent of lung injury in animal models. Notably, reliable
histological assessment of lung injury requires that the lungs be
properly fixed at functional residual capacity, which can be
achieved by inflating the lungs with a pressure of 1520 cm H2O.
Other Considerations

An important consideration is that animal models, because of

their very reductionist nature, are typically used to investigate
one specific aspect of lung injury (e.g., mechanisms of leukocyte
activation, endothelial or epithelial injury, acid-induced lung
injury) in contrast to the human situation where there are often
multiple inciting insults, comorbid conditions, and therapeutic
interventions that all contribute to the clinical picture. Thus,

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although animal models of ALI should reflect one or more of

the clinical features observed in humans, no single animal
model can encompass all of them. On the other hand, the more
restricted the animal model is (i.e., possessing fewer features of
the human situation), the less representative it is of human
disease.
Given these considerations, how can an investigator decide
on an appropriate model system, and which features of ALI are
required for it to be a valid model? The primary consideration in the choice of an animal model of ALI should be the
experimental question to be addressed. For example, if the
primary question pertains to sepsis-induced ALI, then models
incorporating elements of bacterial infection such as intraperitoneal or intravenous administration of lipopolysaccharide or
live bacteria, or cecal ligation and perforation, should be
considered and the relevant endpoints assessed (14). If the
primary question pertains to the mechanisms of lung injury
induced by mechanical ventilation, then various modes of
injurious mechanical ventilation can be imposed on the experimental animals in the presence or absence of other inciting
stimuli such as sepsis, hypotension, or acid-aspiration. If the
primary question relates to the mechanisms of leukocyte-induced
endothelial or epithelial injury, then inciting stimuli can be
given intravenously (e.g., complement activating agents) or via
the airways (chemoattractants, lipopolysaccharide, or hyperoxic
gas mixtures). Some challenges imposed on the animal model
(e.g., extremely large tidal volumes in certain models of
ventilator-induced lung injury or massive doses of lipopolysaccharide in certain models of sepsis) may be so extreme as to be
nonrepresentative of the range of conditions present in humans
with ALI/ARDS, but may be useful to answer very specific
scientific questions. Finally, models in which more than one
inciting stimulus for ALI is present are probably more reflective
of the human situation in which a single inciting stimulus is
rarely present (two-hit hypothesis).
The question remains as to what constitutes the minimal
criteria for the diagnosis of ALI in animal models. Although
there is no consensus about this issue (indeed, this is one of the
primary purposes of this work), several general comments can be
made. First, models in which only one aspect of lung injury is
prominent (e.g., excess neutrophils in the alveolar space without
evidence of physiological pulmonary dysfunction or increased
permeability of the alveolocapillary membrane) are not reflective
of the human situation and should probably not be labeled as
ALI but may still be appropriate to answer specific scientific
questions. Second, depending on the specific features of the
model, it is possible that there may be a divergence between
different aspects of lung injury. For example, under certain
experimental conditions it is possible to induce large numbers
of neutrophils to migrate into the alveolar space with minimal or
only transient evidence of physiological pulmonary dysfunction
or increases in permeability of the alveolocapillary membrane.
Thus, it is prudent to use more than one independent measurement that reliably assesses lung injury in any model of ALI.

III. METHODOLOGY
A series of targeted questionnaires was transmitted by e-mail to
a panel of experts. We requested participation from a total of 29
individuals selected with the goal of generating an international
panel of investigators with broad expertise in lung injury. Of these
29 individuals, two declined, three did not reply, and two failed
to return conflict-of-interest statements, leaving the final number
of participants at 22. The first questionnaire was designed to
generate a list of main features (categories) of ALI in animals, as
well as specific measurements (criteria) that can be used to

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determine if a main feature is present. Questionnaire One yielded

70 individual measurements grouped under eight main features.
The results of Questionnaire One were compiled and returned to
the participants who were asked to rank each measurement as 1.0
(very important), 2.0 (somewhat important), or 3.0 (not important). The results were analyzed and the means and SD of each
measurement were calculated. All measurements with a mean
greater than 2 were removed and redundant measurements
were combined, leaving 32 individual measurements grouped
under main features. Of the original nine main features, four
were removed because none of the measurements reached
a mean of two; these four main features were: evidence of
cellular injury, conversion to a fibrotic response, radiological changes, and other. An additional main feature was
clinical evidence of disease; within this feature only one
measurement, rapid onset (within 24 hours), received a score
of two or less; this measurement was incorporated into the
definition of ALI. Next, the panelists were asked to determine
the relevance of each of these measurements to lung injury,
using a scale of 1.0 to 3.0, with 1 being very relevant, 2 being
somewhat relevant, and 3 being not relevant. Of the 32
measurements, 17 were considered very relevant and 15 were
considered somewhat relevant. Finally, in addition to the
questionnaires, there were three in-detail face-to-face meetings
that allowed members of the panel to discuss the results of each
step, including combining redundant criteria. The final results
are summarized below, and the questionnaires (including responses) are shown in the online supplement.

IV. RESULTS: FEATURES AND MEASUREMENTS OF

ALI IN ANIMALS
1. Main Features of Experimental ALI

The main features of experimental ALI in animals are rapid

onset (within 24 h) and:
1.1 Histological evidence of tissue injury
1.2 Alteration of the alveolar capillary barrier
1.3 An inflammatory response
1.4 Evidence of physiological dysfunction
Of these, the most relevant features were considered to be
evidence of tissue injury and evidence of alteration of the
alveolar capillary barrier. The presence of the main features of
experimental lung injury can be established with the following
measurements:
2. Measurements of Histological Evidence of Tissue Injury

Very relevant

3. Measurements of Alteration of the Alveolar

Capillary Barrier

Very relevant
3.1 An increase in extravascular lung water content
3.2 Accumulation of an exogenous protein or tracer in the
airspaces or the extra vascular compartment
3.3 Increase in total bronchoalveolar (BAL) protein concentration
3.4 Increase in concentration of high molecular weight proteins in BAL fluid (e.g., albumin, IgM)
3.5 Increase in the microvascular filtration coefficient
Somewhat relevant
3.6 Increase in lung wet/dry weight ratio
3.7 Translocation of a protein from the airspaces into plasma
3.8 Increased lung lymph flow
3.9 High lymph protein concentration
4. Measurements of the Inflammatory Response

Very relevant
4.1 Increase in the absolute number of neutrophils in BAL
fluid
4.2 Increase in lung myeloperoxidase (MPO) activity or
protein concentration
4.3 Increase in the concentrations of proinflammatory cytokines in lung tissue or BAL fluid
Somewhat relevant
4.4 Increases in procoagulatory activity
4.5 Increased expression of adhesion molecules
4.6 Conversion of the neutrophilic alveolitis into a mononuclear alveolitis with time
4.7 Increase in levels of complement factors and matrix
metalloproteinases
5. Measurements of Physiological Dysfunction

Very relevant
5.1 Hypoxemia
5.2 Increased alveolararterial oxygen difference
Somewhat relevant
5.3 PaO2/FIO2 , 200

2.1 Accumulation of neutrophils in the alveolar or the

interstitial space

5.4 Increase in spontaneous minute ventilation

2.2 Formation of hyaline membranes

5.5 Increase in spontaneous respiratory rate

2.3 Presence of proteinaceous debris in the alveolar space

(such as fibrin strands)
2.4 Thickening of the alveolar wall
2.5 Enhanced injury as measured by a standardized histology
score
Somewhat relevant
2.6 Evidence of hemorrhage
2.7 Areas of atelectasis
2.8 Gross macroscopic changes such as discoloration of the
lungs

To determine if ALI has occurred, we recommend that at least three

of the four main features of ALI be identified. To determine if any
of the main features of ALI are present, we recommend using at least
one of the very relevant measurements listed above, and preferably,
one or two additional separate measurements to confirm the results.
We would like to emphasize that not all of the measurements
listed can or should be performed in every study, and that measurements not included in the list are by no means irrelevant. For
example, there are many measurements that are useful for assessing
lung injury, but can only be performed in specialized laboratories (e.g.,
animal computer tomography). We have tried to include measurements that are within the reach of most laboratories located at major

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universities and health care facilities. We would also like to point out
that, depending on the experimental question, the fact that fully
developed ALI has occurred may not be necessary or relevant to
establish. For example, a model with alveolar neutrophilia
without changes in alveolar permeability is suitable for studying the mechanisms of neutrophil migration, even if such
a model does not reproduce all of the main features of
ALI. Thus, our list of features and measurements of ALI is
intended as a guide for investigators, but ultimately investigators should choose any particular measurement based on their
unique experimental questions and the characteristics of their
experimental design.
One additional aspect of the proposed features is that they
emphasize functional parameters of injury over individual molecular pathways. This highlights an important distinction between evidence of activation of a particular signaling pathway
(e.g., the MAP kinase pathway) and lung injury. In this regard,
there may be activation of (one or multiple) signaling pathways
without concomitant lung injury. Conversely, there may be
evidence of lung injury without activation of particular signaling
pathways. This is not to say that mechanistic studies aimed at
dissecting the role of signaling pathways in lung injury are not
important but rather that the presence or absence of evidence of
activation of these pathways is distinct from whether or not
there is evidence of ALI.

V. PRACTICAL ASPECTS OF MEASURING

ALI IN ANIMALS
1. Measuring Histological Evidence of Tissue Injury

Histological evidence of tissue injury was identified as the most

relevant defining feature of ALI. The normal lung is characterized by thin alveolar walls with occasional intra-alveolar macrophages and vanishingly few neutrophils (Figure 1A21B).
Commonly, during the dehydration process associated with the
preparation of tissues, the peribronchial space may appear
expanded (Figure 1A). This finding must not be confused with
peribronchial edema. The intratracheal administration of injurious substances is usually associated with patchy injury, and
large areas of the lung may be spared (Figure 1C). To facilitate
the identification of areas that have been exposed to the instillate, colloidal carbon (1:100 dilution) can be added to the
instillate; the colloidal carbon will be phagocytosed by resident
macrophages and appear as intracellular black dots (Figure 1).
This can be particularly useful when a negative experiment
raises doubts as to whether the instillate could have been
inadvertently administered into the esophagus. Some types of
injury are primarily associated with neutrophil infiltration and
deposition of proteinaceous debris or fibrin strands in the
airspaces but with normal alveolar walls (Figure 1D). In
contrast, other types of injury may be associated with marked
thickening of the alveolar walls, which may contain abundant
neutrophils but no evidence of intra-alveolar neutrophil infiltrates or protein deposition (Figure 1E). Hyaline membranes,
when seen, appear as pink deposits on the alveolar walls
stained with hematoxylin and eosin (Figure 1F). It is important
to note that the presence of increased neutrophil numbers in
the BAL fluid is unlikely to be associated with obvious intraalveolar neutrophil infiltrates unless the total BAL neutrophil
counts are greater than 106, because the BAL procedure collects
neutrophils from the entire lung. Also, in certain animals the
neutrophils may display light staining; for example, in rabbits
neutrophils are slightly eosinophilic and therefore not strictly
a neutrophil in the sense of a cell that does not stain (Figure
2C22D).

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The Committee identified the need for a scoring system to

quantify the extent of histologic lung injury. Any scoring system
must be approached with caution, however, as it may not
function similarly across different model systems, the sensitivity
is limited, and there may be considerable interobserver variation. It is therefore not surprising that the scoring of injury in
the research setting has met with limited success despite
a number of equally valid approaches. Furthermore, appropriate scoring of tissue sections requires a considerable amount of
time, including the period required to validate the system. The
simplest tissue injury scoring system is a binary approach in
which sections are categorized as either injured or normal. A
number of more complex systems have been used in the
literature, each incorporating features of ALI tissue to variable
degrees. The use of strict morphometric criteria maximizes the
reproducibility of the data, but adherence to strict morphometric standards is difficult to achieve in many laboratories. In this
section, we propose a histologic ALI scoring system incorporating criteria that are accessible to both laboratory researchers
and pathologists (Table 1). To mitigate against interobserver
variability, we constructed a relatively simple scheme that uses
coarse gradations of severity for each separate finding, which
differs from previous scoring systems (15, 16).
Slides for evaluation should be prepared from suitably inflated
(1525 cm H2O), formalin-fixed paraffin-embedded tissue that has
been stained with hematoxylin and eosin. Due to the patchy
nature of ALI, at least 20 random high-power fields (4003 total
magnification) should be independently scored in a blinded fashion for each condition. The selection of random fields typically
involves successive random displacements (each at least one highpower field in length) from the current position. More rigorous
sampling may use a random function to generate (x, y) coordinates
on the slide. Sophisticated strategies, such as stratified sampling
and Poisson-disk distributions, may allow for more even sampling
of the tissue and reject overly close samples. Furthermore, at least
50% of each field should be occupied by lung alveoli; fields that
consist predominately of the lumen of large airways or vessels
should be rejected. Each of five histological findings is graded
using a three-tiered schema summarized in Table 1 and outlined
in detail below.
Neutrophils are counted in the alveolar airspaces, alveolar
walls, and interstitium. Alveolar wall neutrophils include cells
that have entered the interstitium as well as those that are
circulating or adhere to the alveolar capillaries. It should be
noted that, as mentioned earlier, the neutrophils of some
animals differ significantly in morphology from those in
humans. For instance, murine models typically show ring-form
neutrophils, and rabbit neutrophils appear eosinophilic on
a Wright-Giemsa stain (i.e., they look pink rather than neutral
in color). If neutrophils are not visible within the field, a score
of zero is recorded for that field. One to five neutrophils
give a score of one, and more than five neutrophils give a score
of two.
Hyaline membranes, the hallmark of ALI in humans, are not
routinely observed in many animal systems, most notably in
murine models. The presence of even a single well-formed
eosinophilic band of fibrin within the airspace earns a score of
one, whereas multiple membranes visible in the field are scored
as two. Pink proteinaceous debris filling the airspace is considered in an analogous manner to hyaline membranes.
The evaluation of septal thickening is highly dependent on
the uniformity of inflation throughout the lung. If regions are
underinflated, either due to errors in technique or variations in
airway geometry, septae may appear artifactually thickened.
Therefore, only septal thickening that is equal or greater than
twice normal is considered in these criteria. Furthermore, septal

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Figure 1. Normal and injured lungs. (A, B) Normal mouse

lungs stained with HandE (A, 2003; B, 6303). The
alveolar walls are very thin and the alveoli contain
occasional alveolar macrophages. Note that the artifactual
expansion of the peribronchial tissue (asterisk), which
commonly results from tissue processing, does not represent edema. A, airspace; V, blood vessel; TII, type II cell;
M, alveolar macrophage; AW, airway. (C and D) show
patchy neutrophilic infiltrates with deposition of fibrin
strands (arrows in D). Note that the alveolar walls are thin.
(E ) shows thickened alveolar walls with intramural neutrophils (arrows). Note the absence of neutrophils inside
the alveoli. The black substance inside the macrophages is
colloidal carbon. (F ) shows hyaline membranes (arrows).

thickness should not be evaluated in alveolar septa that are

directly adjacent to a blood vessel or airway because these are
normally thickened by the collagen present in the peribronchovascular bundle.

To generate a lung injury score, the sum of each of the five

independent variables shown in Table 1 are weighted according
to the relevance ascribed to each feature by the Committee, and
then are normalized to the number of fields evaluated. The

Figure 2. Comparison of neutrophils from three mammalian species. (A) Human neutrophils show a typical
multi-lobed nucleus interconnected by thin chromatin
strands, and a colorless cytoplasm on Wright-Giemsa
stains. Notice the presence of two eosinophils (open
arrows) that can be distinguished by their pink cytoplasm
and bilobulated nuclei (peripheral blood cytospin, DiffQuick, 4003). (B) Murine neutrophils exhibit characteristic ring- or pretzeloid-shaped nucleus with drumstick
shaped nodules containing pericentric heterochromatin;
as in humans, the cytoplasm is colorless, whereas eosinophils are pink and have a bilobulated nucleus (peripheral
blood cytospin, Diff Quick, 4003). In contrast, (C and D)
rabbit neutrophils are notorious for their pink coloration
(black arrows in D), and can be easily confused with the
eosinophil of other mammalian species; they can be
recognized because of their multilobulated nucleus, which
is very similar to that of the human neutrophil (C, lung
from a rabbit with ventilator-induced lung injury, HandE,
6303; and D, further magnification from the section
in C ). Note the different color of a macrophage (D,
thick black arrow).(A) and (B) are courtesy of Dr. Jatinder
Juss, University of Cambridge, UK; (C ) and (D) are
courtesy of Dr. G. Matute-Bello, University of Washington,
Seattle, WA.

American Thoracic Society Documents

731

TABLE 1. LUNG INJURY SCORING SYSTEM

Score per field
Parameter
A. Neutrophils in the alveolar space
B. Neutrophils in the interstitial space
C. Hyaline membranes
D. Proteinaceous debris filling the airspaces
E. Alveolar septal thickening

none
none
none
none
,2x

15
15
1
1
2x4x

.5
.5
.1
.1
.4x

Score 5 [(20 3 A) 1 (14 3 B) 1 (7 3 C) 1 (7 3 D) 1 (2 3 E)]/(number of

fields 3 100)

resulting injury score is a continuous value between zero and

one (inclusive).
2. Measuring Alterations in Permeability

There are several potential approaches for measuring changes

in lung vascular and epithelial permeability (17, 23). In large
animals, such as sheep and goats, it is possible to collect lung
lymph and measure the concentration of total protein in the
lymph and in the plasma. The normal ratio is approximately
0.50 (24). If the left atrial pressure increases, then the lymph to
plasma total protein ratio declines, because more salt and water
are being filtered out of the lung capillaries than protein. If an
animal is given an injury that increases permeability, such as
oleic acid, endotoxin, live bacteria, or acid, then the lymph-toplasma total protein ratio will normally not change. The lung
lymph flow will increase, but the maintenance of a normal
lymph-to-plasma total protein ratio indicates that the mechanism for the increase in fluid filtration is primarily increased
permeability. The one caveat is that an increase in cardiac
output with an increase in the surface area of lung perfusion will
result in an increase in lung lymph flow with no change in
lymph-to-plasma total protein ratio, so this could be misinterpreted as an increased permeability when it is really higher
pulmonary blood flow with greater surface area of perfusion.
Another experimental approach that is useful for the isolated perfused lung is to measure the microvascular filtration
coefficient, which can be interpreted as reflecting a change in
lung vascular permeability. However, it is important to realize
that the commonly utilized gravimetric measurement procedure
depends on several assumptions such as an isogravimetric state
(weight equilibrium) before and after the applied increment in
hydrostatic pressure, the absence of alveolar edema and a constant vascular surface area (25). If these conditions cannot be
met, it may be more appropriate to use the phrase apparent
filtration rate. Thus, to calculate a filtration coefficient appropriately, the vascular filling component must be excluded from
the analyses, applied pressure increments should be small,
alveolar edema should be avoided, and lymph flow should be
excluded.
In terms of smaller animal studies (rats, rabbits, and mice),
a precise way to quantify an increase in lung vascular and
epithelial permeability is to use a protein labeled with a radioactive isotope such as 125I-albumin or 131I-albumin, fluorescentlabeled albumin, or a fluorescent-labeled high molecular weight
dextran, and then to demonstrate that there is accumulation of
this tracer in the extravascular compartments of the lung by
either (1) homogenizing the intact lung and subtracting the
blood volume or by (2) measuring the accumulation of this
labeled protein in the distal airspaces of the lung using
bronchoalveolar lavage (BAL). Experimental interventions that
reduce permeability should result in a reduced quantity of
labeled protein accumulation in the airspaces and/or the
extravascular compartments of the lung. It is important to

verify that the label has remained bound to the protein or

dextran molecule. The possibility of dissociation of the label is
particularly important when using albumin bound to radioactive
iodine. This can be determined by actively dissociating the
isotope from the albumin using 20% trichloroacetic acid and
then comparing the total free radioactivity with the radioactivity of the untreated sample (26). Also, the permeability of the
alveolar epithelium can be measured by instilling a labeled
protein or dextran into the air spaces and measuring its
accumulation in the plasma and its loss from the lung. One
emerging problem with albumin-based markers is transport by
transcytosis, which may lead to increased tissue albumin in the
absence of increased permeability (27).
A popular method of measuring changes in capillary permeability involves the use of Evans Blue dye, a fluorescent azo dye
that was originally described in the 1930s as a tool to determine
blood volume using fluorometry (28). Evans Blue dye was
subsequently used to determine changes in capillary permeability in tissues (29). The method requires the intravenous injection of Evans Blue dye, followed by determination of the
fluorescence of formamide-treated lung extracts at excitation 5
620 nm, emission 5 680 nm (30, 31). The determination of the
Evans blue dye concentration (EBD) in a sample should include
a correction for contaminating heme pigments using the formula E620(EBD) 5 E620 2 (1.426 3 E740 1 0.030) (30).
Evans blue dye is commonly used because it allows for the
determination of permeability changes without the use of
radioactive isotopes. This is similar to fluorescently labeled
dextrans, although the later have the advantage that multiple
sizes of dextran can be selected and the amount of processing
required is minimal.
A crude method to determine increased lung water is to
calculate wet-to-dry ratios. The wet lung is weighed, and then
placed in an oven and weighed daily until its weight stabilizes
(dry weight). The wet-to-dry ratio then becomes an estimate of
the total water content of the lung. One problem with this
method is that with very small lungs, small errors in weight
measurement may result in a large variability in the ratios, so
particular care should be exercised when measuring the lung
weight.
Another approach is to measure the total protein concentration in the BAL, which should reflect the accumulation of
extravascular protein primarily from an increase in permeability. In this case, the total quantity of protein is determined by
multiplying the concentration of the protein in the BAL fluid by
the volume of the lavage. Hydrostatic stress will displace some
intravascular protein into the interstitial and alveolar compartments, so if needed, controls can be done with elevated left
atrial pressure or hydrostatic stress to show that the quantity of
protein under these control hydrostatic conditions is less than
under conditions of increased permeability. Furthermore, the
BAL total protein concentration can be influenced by the
protein derived from inflammatory and lung epithelial cells that
are present in the alveolar space, or by changes in the ability of
the alveolar epithelium to clear water from the airspaces (28, 29,
31). For instance, the alveolar protein concentration can increase dramatically in nonperfused lungs (30). It is possible to
test for the presence of a very high molecular weight molecule
such as IgM (900kD) as a marker of increased lung permeability. As BAL measurements are notoriously difficult to standardize, we recommend that the recovery of the BAL fluid be
reported.
The collection of pulmonary edema fluid from animals can
also serve as a useful measurement (32). If edema fluid is
collected in the early phase (first 30 min) after the development
of experimental or clinical pulmonary edema, then the concen-

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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 44 2011

Figure 3. Neutrophils are readily identified by their

nuclear morphology. A composite of cells types found in
the alveolar space and identified by differential staining
with Wright-Geimsa. Neutrophils (black arrowheads) are
identified by their segmented nuclear morphology. Alveolar macrophages (white arrowheads) are identified by
their large size and high cytoplasm to nuclear ratio. The
cytoplasm of alveolar macrophages can also be quite
granular. Lymphocytes (yellow arrowheads) are identified
by their prominent nuclear staining with little cytoplasm.
Magnification 31,000 under oil.

tration of total protein in the edema fluid can be compared with

time-matched plasma samples. If the ratio is greater than 0.65,
then it is likely to reflect an increase in permeability. If the
edema fluid sample is collected more than 1530 minutes after
the onset of alveolar edema, then reabsorption of alveolar fluid
may occur, resulting in concentration of the alveolar protein
and providing a misleading result regarding the mechanism of
the pulmonary edema. For example, if hydrostatic pressure is
raised sufficiently to cause hydrostatic edema with alveolar
flooding, then the initial concentration of alveolar protein will
be less than 65% of plasma protein. However, if the elevated
hydrostatic pressure is returned to normal, then reabsorption of
salt in the water will occur rapidly, driven by active ion
transport across the lung epithelium and resulting in a elevated
concentration of alveolar protein, which, when compared with
the concentration of that protein in plasma, will suggest that
there is an underlying increase in permeability when in fact this
is not the case.
The measurement of extravascular lung water is valuable in
studies of mechanisms of increased permeability pulmonary
edema. However, it should be recognized that an increase in
extravascular lung water alone does not categorize the lung
edema as hydrostatic or increased permeability edema. Also,
investigators should remember than an increase in lung vascular
pressure can markedly increase the extent of pulmonary edema
in the face of an increase in lung vascular permeability so that two
mechanisms for edema formation may occur at the same time.
3. Measuring Inflammation

ALI is most frequently accompanied by an inflammatory response within the lungs. Among possible measurements of
inflammation, three were identified by the panel as very
relevant. These included total neutrophil counts in the BAL
fluid, lung myeloperoxidase (MPO) (a surrogate for neutrophil
influx), and measurements of proinflammatory cytokines. Each
of these parameters is easily measured in most research
laboratories by the following methodologies.
Neutrophils in the BAL uid. The quantification of neutrophils in BAL fluid reflects migration of neutrophils into the
airspaces of the lungs. This measurement is most readily
accomplished by performing a differential analysis of cells
collected from the BAL. The addition of ethylenediamine
tetraacetic acid (EDTA) to the fluid used to perform the
BAL (e.g., 0.9% NaCl or PBS) will prevent clumping of the
cells and facilitate counting. After collection, the samples
should be kept on ice and analyzed as soon as possible to
prevent cell death. Animals can be killed at relevant time points
(e.g., 1, 3, 5, and 7 d postinsult), the trachea can be cannulated
and saline (generally a volume of 1 ml for mice) is instilled into
lungs and then recovered. This procedure can be repeated
several times. The total cell pellet is then collected by centri-

fugation and cells are sedimented by centrifugation (cytospun)

onto glass slides and stained with Wright-Giemsa or hematoxylin and eosin. Neutrophils are identified using light microscopy
by their characteristic nuclei (Figure 3). A minimum of 300 total
cells should be counted to obtain a reliable neutrophil percentage in the BAL. This percentage can be multiplied by the total
cell numbers recovered to arrive at an absolute count in the
BAL fluid. It is always preferable to report the total number of
neutrophils in the BAL fluid, along with, or instead, of the
percentages. This is because a decrease in the percentage of
BAL neutrophils in response to a given treatment may reflect
an increase in the number of macrophages, without any real
change in the number of total neutrophils. Alternatively,
neutrophils can be readily identified in the BAL fluid by flow
cytometry using specific antibodies against Ly6G (33, 34).
Neutrophils, being granular, have a characteristic side versus
forward scatter profile, which is evident using this technique.
Myeloperoxidase (MPO) activity. MPO is a glycoprotein
expressed in all cells of the myeloid lineage, but found most
abundantly in azurophilic granules of neutrophils (35). MPO is
released by activated neutrophils and, as such, measurement of
MPO in cell-free BAL fluid serves as a surrogate for the
accumulation of activated neutrophils in the airspaces of the
lung. Alternatively, measurement of MPO in whole lung
homogenates reflects accumulation of neutrophils in the lungs,
and as such may be a useful complement to the measurement of
neutrophils in the BAL, which reflects migration of neutrophils
into the airspaces. MPO can be measured effectively either by
commercial ELISA kits according to manufacturers directions
or by a colorimetric assay which correlates with MPO activity as
described previously (33, 36, 37).
When MPO activity is measured in whole lung supernatants,
it is important to add a buffer that is designed to preserve the
activity of the enzyme (e.g., see Reference [38]). The determination of MPO activity may be associated with greater variability than the ELISA methods.
Concentrations of proinammatory mediators. ALI is almost invariably associated with elevations in proinflammatory
mediators including tumor necrosis factor (TNF), IL-1, IL-6,
macrophage inhibitory factor (MIF), transforming growth factor (TGF), platelet activating factor (PAF) and the leukotriene
LTB4 (reviewed in [3943]), although anti-inflammatory cytokines such as IL-10 are usually also elevated (44). In humans,
IL-8 is a prominent neutrophil-recruiting chemokine. The
murine orthologs of this molecule are KC (CXCL1) and MIP2 (CXCL2) (39, 45). These mediators are readily measured by
commercial ELISAs or multiplex assays using either BAL fluid
or lung homogenates. Measurement of LTB4 in biological fluids
is best accomplished by first extracting the lipids using C18
cartridges from the biological sample, drying them under
nitrogen, and then performing the ELISA on the reconstituted

American Thoracic Society Documents

sample (46). Mediators can also be evaluated using real-time

PCR assays to look for mRNA expression, or in the case of
LTB4, for the up-regulation of the 5-lipoxygenase (5-LO), 5-LO
activating protein, or LTA4 hydrolase enzymes involved in the
lipid synthesis.
Particular care should be exercised to prevent degradation of
cytokines. This can be accomplished by adding commercially
available protease inhibitors to the samples. In addition, some
cytokines such as TNF are extremely sensitive to freeze and
thaw cycles; accordingly, we recommend storing the samples in
individual aliquots and performing measurements after a single
thaw. When using bead-based multiplex assays on tissue homogenates, the addition of DNase to the samples will decrease
viscosity and prevent clogging of the machine.
Cytokine mRNA expression using quantitative PCR or
expression arrays can be used to identify patterns of cytokine
gene expression. This is a powerful technique, but requires
particular attention to three key issues. The first issue is that
RNA is easily degraded, and it is essential to ensure that sample
collection strictly adheres to protocols designed for RNA
preservation; this is particularly important when the samples
are intended to be used for microarray analysis (47, 48). The
second issue is the selection of the reference gene. There are
increasing concerns that commonly used reference genes such
as GAPDH and actin have variable expression under certain
conditions, and while other genes such as HPRT and 18S RNA
appear to display more stable expression, there is no consensus
as to the preferred gene to use (4951) Finally, mRNA
expression does not necessarily correlate with protein expression, and key findings should be verified by measuring the actual
protein.
Additional criteria that were considered somewhat relevant
for the measurement of ALI included measurements of procoagulant activity (52), expression of adhesion molecules (53),
conversion of the neutrophilic alveolitis to a monocytic alveolitis with time, and elevations in the levels of specific complement factors (54) and matrix metalloproteinases (55).
4. Physiological Dysfunction

Measurements of gas exchange. The main purpose of the lung

is to absorb oxygen and remove carbon dioxide. As such, it is
noteworthy that hypoxemia and an increased alveolararterial
oxygen difference [(Aa)DO2] were listed as the most relevant
measures of physiological dysfunction in models of lung injury.
Despite this, it is important to point out that hypoxemia is not
a direct measure of injury per se, but is often a manifestation of
injury. Hypoxemia can occur due to many physiological mechanisms not associated with ALI (see below). Thus, measurement of oxygenation should not be the only variable that is
assessed in determining if an animal has lung injury, but
measuring oxygenation can be very useful in helping to assess
the degree and time course of injury.
Hypoxemia and alveolararterial oxygen difference [(A-a)
DO2]: Hypoxemia can be assessed by measurement of the
arterial partial pressure of oxygen (PaO2) from arterial blood
gases, or from oximetry; typically, hypoxemia is defined as PaO2
, z60 mm Hg, or SpO2 , 90%. Measurement of arterial blood
gases are usually more accurate and have the added advantage
of providing a value for PaO2, which is important in assessing the
extent to which any decrease in PaO2 is due to hypoventilation
(increased PaCO2). This can be calculated using the (simplified)
alveolar gas equation [PAO2 5 (PB-PH2O)
FIO2 PaCO2/R],
where PaO2 is the alveolar partial pressure of oxygen, PB is the
atmospheric pressure, PH2O is the saturated vapor pressure of
water, FIO2 is the inspired fraction of oxygen, PaCO2 is the
arterial partial pressure of carbon dioxide, and R is the re-

733

spiratory quotient. The alveolararterial oxygen difference

[AaDO2 5 (PAO2 PaO2)] can be used to correct for differences
in PaCO2, but can vary substantially with FIO2 depending on the
degree of ventilation/perfusion (V/Q) mismatch versus shunt.
As such, if one wants to compare oxygenation at different time
points and/or among animals, the same FIO2 (often chosen at an
FIO2 of 0.5 or 1.0) should be used.
As can easily be deduced by the alveolar air equation,
oxygenation also varies with barometric pressure, and respiratory quotient. Taking into account the barometric pressure is
important if one is doing experiments at a high altitude or
comparing results in laboratories at different altitudes.
Arterial oxygenation can vary substantially with a number of
physiological variables, many of which are controlled by the
investigator. In animals with ALI, oxygenation may vary with
changes in mean airway pressure, which is dependent upon the
inspired fraction of oxygen (FIO2), the level of positive endexpiratory pressure (PEEP), tidal volume, and the inspiratory
to expiratory ratio. Oxygenation can also vary with changes in
the stiffness of the chest wall or in body position (e.g., prone vs.
supine). As such, depending on the purpose of the measurement, estimates of oxygenation should be made by keeping as
many of these variables as constant as possible.
In animals that are being mechanically ventilated, oxygenation
can also depend on lung volume history, (i.e., the size of the
breaths preceding the oxygen measurement), especially in models
of injury characterized by substantial atelectasis. One approach
that is used to mitigate this effect is to provide a relatively
uniform volume history by performing a recruitment maneuver
immediately before measurement of the oxygenation variable(s).
If there is significant pulmonary shunt, arterial oxygenation
can decrease with decreases in cardiac output because of
a decrease in mixed venous oxygenation. This is important if
one is using arterial oxygenation as a measure of the degree of
lung injury, because a decreasing PaO2 may not necessarily
signify a worsening of lung injury but may signify a worsening in
hemodynamics.
The PaO2/FIO2 ratio (P/F) is used to define (along with other
characteristics) human acute respiratory distress syndrome
(P/F , 200 mm Hg) and ALI (P/F , 300 mm Hg), based on the
American European Consensus Committee. There are no
agreed-upon diagnostic criteria of different lung injury entities
(ALI vs. ARDS) in animal models of lung injury, and indeed
this workshop proposed that the PaO2/FIO2 ratio should only be
considered somewhat relevant for assessing physiological
function in animal models of ALI. This may be because the
Committee members felt that it is easier to make measurements
under more identical controlled FIO2 in animal models, in which
case the alveolararterial oxygen difference would suffice.
Measurement of arterial blood gases are reasonably easy to
measure in relatively large animals but may be quite difficult to
measure in very small animals (e.g., mice), especially repeatedly
during the course of an experiment. As such, the workshop
participants felt that minute ventilation and respiratory frequency might be variables that are somewhat relevant in
assessing lung injury. These variables diverge substantially
among species of different sizes, which may not be an issue in
comparisons to studies on the same species. Most importantly,
they are affected by a number of factors that may bear no
relationship to ALI, including the degree of sedation, pain,
body temperature, acidbase balance, and so forth.
Some general considerations. Animal studies are fraught
with high variability in the results, and this is true even under
strictly controlled conditions and when all the animals are
genetically identical. One common source of variability is
animal stress, and this is particularly important in mice, which

734

AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 44 2011

TABLE 2. PRESENCE OF VERY RELEVANT CRITERIA IN STANDARD MODELS OF ACUTE LUNG INJURY

Histological evidence of tissue injury

Accumulation of neutrophils in the alveolar/interstitial space
Formation of hyaline membranes
Proteinaceous debris in alveolar space
Thickening of the alveolar wall
Injury by a standardised histology score
Alteration of the alveolar capillary barrier
Increased extravascular lung water content
Accumulation of protein/tracer in airspaces/extravascular space
Total BAL protein concentration
BAL concentration of high molecular weight proteins
(Micro-) Vascular filtration coefficient (Kf)
Inflammation
BAL total neutrophil counts
Lung MPO activity
Concentrations of cytokines
Physiological dysfunction
Hypoxemia
Increased A-a oxygen difference

VILI

LPS

Bacteria

1
1
1
1
1

1
1
1
1
1

1
1
1
1
1

1
1
1
1
1

1
1
1
1
(1)

1
1
1
1
(1)

1
1
(1)

1
1
1

1
1
1

1
1

1
1

1
1

O2

OA

Acid

I/R

1(1)
1(4)
1(1)
1(8)
1(10)

1(1)
1(5)
1(1)
1
1(11)

1(2)
1(2)
1(7)
1(9)
1(2)

1(1)
1(10)
1(16)
1(8)
1(21)

1(12)
1(14)
1(12)
1(5)
1(12)

1(13)
1(13)
1(2)
1(18)
1(2)

1(1)
1(1)
1(1)

1(16)
1(16)
1(16)

1(12)
1(16)
1(5)

1(22)
1(13)
1(13)

2(23)

1(1)
1(1)

1(16)
1(16)

1(9)
1(9)

1(1)
1(3)
1(6)
1(3)
1(3)
1(1)
1(1)
1(15)
1(17)
(1)(19,20)*

Definition of abbreviations: Aa, alveolar-arterial; BAL, bronchoalveolar lavage; I/R, ischemia-reperfusion; MPO, myeloperoxidase; OA, oleic acid; VILI, ventilatorinduced lung injury.
1 or (1), the criterion is present in virtually all or the majority of studies using this model.
2, criterion is absent.
* 12 hours at 100% O2 increased lung capillary permeability as assessed by lymphatic canulation in dogs (19), whereas 1 hour at 100% O2 did not further increase Kf
in a VILI model 2 in rabbits (20).

on continued hyperoxic ventilation at 100% O2 for 96 h; in rabbits, 4 h at 50% O2, but not 36 h at 100% O2 reduced PaO2/FIO2 (24) (130); in baboons, 96 h at
100% O2 caused a progressive decline in PaO2/FIO2 (23).

may be affected by even minor changes in light/dark cycles or

by minor handling. Care should be taken to ensure that sources
of stress, including minor handling, are reduced to a minimum,
and that control and experimental animals are all handled in
exactly the same way. In addition, the performance of animal
studies is almost always associated with a learning curve, and
even very experienced technicians will be better at specific
techniques toward the end of a study. It is extremely important
to avoid working with all the experimental animals first and all
the controls at the end of a study; instead, controls and
experimental animals should be alternated throughout the study
and if at all possible, the investigators should be blinded to the
experimental conditions. Study protocols should ensure humane
treatment of all animals, including the use of protocolized
monitoring to identify evidence of animal discomfort, the use
of clear criteria to minimize discomfort by providing sedatives
or anesthesia, and the use of prospective criteria to identify
animals that should be subject to early euthanasia. All animal
studies should only be performed after approval by the investigators Institutional Animal Care and Use Committee
(IACUC) or equivalent regulatory body.

VI. CRITICAL ASSESSMENT OF SELECTED COMMON

MODELS OF LUNG INJURY
We evaluated the presence of criteria considered as very
relevant in selected common models of lung injury based on
representative studies from the literature. A literature search
over the last five years ranked mechanical ventilation with
high tidal volumes or high peak inspiratory pressures, pulmonary or systemic administration of endotoxin, and inhalation
or instillation of live bacteria as the three most commonly
applied models of lung injury in animals. Evidence of the
main features of ALI by the defined measurements was
analyzed for these three models, as summarized in Table 2.
A more extensive review of animal models of lung injury is
beyond the scope of this manuscript, but can be found
elsewhere (7).

Ventilator-Induced Lung Injury

Ventilator-induced lung injury (VILI) models typically present

characteristic features of tissue injury such as interstitial
thickening and alveolar infiltration of neutrophils (56). Hyaline membranes are seen in larger animals, but are rare in rats
or mice unless the tidal volumes are very elevated (Figure 1F:
rat lungs ventilated for 60 minutes with tidal volumes 5 35
cc/kg and PEEP 5 0). Tissue injury is also documented by
blinded assessments of histopathological scores based on edema severity, hemorrhage, septal thickening, and interstitial or
alveolar infiltration of inflammatory cells in different species
ventilated with high tidal volumes of 2030 ml/kg body weight
for several hours (57, 58). VILI also causes prominent changes
in water and protein permeability as evidenced in isolated rat
and mouse lungs by increased wet-to-dry weight ratios and
microvascular filtration coefficients (59, 60). In vivo, ventilation
with tidal volumes of 20 to 30 ml/kg body weight results in an
accumulation of Evans bluelabeled albumin in the lung or
the extravasation of intravascular radiolabeled plasma proteins
such as albumin or transferrin into the extravascular space (57,
61, 62). Increased concentrations of albumin and total protein
are detectable in the BAL (63, 64). Recruitment of inflammatory cells as assessed by lung MPO assay or BAL neutrophil
counts is generally evident (58, 64). Increased formation and
release of cytokines from overventilated lungs has been reported
in mouse and rat studies using isolated lung preparations, but
may not be present in all models of VILI (30, 65, 66). Last, but
not least, over-ventilation causes a steady decline in arterial
oxygen saturation which typically results in severe hypoxemia
within several hours (56, 67).
Endotoxin-Induced Lung Injury

Endotoxin challenge by inhalation, intratracheal instillation,

or intravenous infusion of various lipopolysaccharides typically results in considerable tissue injury, which can be evident
within less than 1 hour and is characterized by neutrophil
accumulation in the alveolar and interstitial space, alveolar

American Thoracic Society Documents

wall thickening, and accumulation of proteinaceous edema

and detritus in the alveolar space (34, 6872). Endotoxindependent tissue injury has also been confirmed by semiquantitative histological scores based on signs of inflammation
and hemorrhage in the alveolar and interstitial space, edema,
atelectasis, hyaline membranes, and cellular necrosis (70, 72,
73). Increased wet-to-dry weight ratios, Evans blue accumulation in the lung, and increased albumin and total protein
concentrations in the BAL fluid after endotoxin challenge
demonstrate acute alterations in water and protein permeability (70, 7377). Of note, changes in lung vascular filtration
coefficient (Kf) are present in some but not all models of
endotoxin-induced lung injury. Increased Kf values were
detected after endotoxin challenge in vivo (78) and also after
infusion of LPS in buffer-perfused isolated rat and piglet lungs
(79, 80), but other studies found increased Kf only in serumperfused but not in plasma-perfused isolated rat lungs (81),
whereas no changes were detected in goat lungs perfused
with autologous blood (82). In blood-perfused rat lungs,
endotoxin only increased microvascular permeability in the
presence of a nitric oxide synthase inhibitor (83). Increased
neutrophil counts in the BAL, high MPO activity, and lung
cytokine levels are hallmarks of the inflammatory response in
virtually all models of endotoxin-induced lung injury (72, 73,
76, 77, 79, 84), whereas hypoxemia is a less consistent finding
(69, 70, 84, 85).
Lung Injury by Live Bacteria

Microbial infection of the lungs by inhalation of aerosolized

bacteria or direct intranasal, endotracheal, or endobronchial
instillation is used extensively as an experimental model of
pneumonia in animals (86). Tissue injury in this model is
striking with accumulation of neutrophils in the alveolar and
interstitial space but also includes alveolar wall thickening
and hyaline membrane formation (8694); this can be assessed
semi-quantitatively by histological scores. Permeability is
commonly increased as demonstrated by elevated wet-to-dry
weight ratios, leakage of radioactive protein tracers from the
vascular space or accumulation of Evans blue dye, increased
protein levels in the BAL, and elevated vascular filtration
coefficients (88, 90, 93, 95100). Yet, in individual cases, for
example, in a mouse model of intratracheal challenge with E.
coli, changes in the filtration coefficient and lung weight gain
have been reported to be minimal over a period of up to 12
hours (92). Inflammation markers, that is, increased neutrophil counts in the lavage, MPO activity and cytokine levels, as
well as systemic hypoxemia, are in general uniformly present in
lung injury that is induced by live bacteria (88, 92, 97, 99101).
Taken together, the most frequently applied standard
models demonstrate the main features of ALI described in this
article. Yet, not all criteria are consistently met by all models. It
is therefore important to note that failure to comply with
a single criterion does not necessarily compromise the validity
of an established model.

VII. LIMITATIONS
This document represents a consensus reached among a diverse
group of investigators actively involved in the study of ALI.
However, it does not reflect unanimity, and there were clearly
areas of diverging opinions indicating the complexities involved in many of these measurements. As such, this document
is not meant to be prescriptive or to suggest that there is only
one way of assessing ALI, insofar as different model systems
and local availability of instrumentation may be the overriding
factors that dictate the variables to be measured. As discussed

735

above, it is prudent to measure several independent variables

that reflect various aspects of ALI rather than relying on
a single measurement. Finally, a number of the recommendations in this report were made despite a paucity of data. As
such, we used an iterative process to reach consensus and
leave open the possibility that future studies may refute these
recommendations.

VIII. SUMMARY AND CONCLUSIONS

In summary, we present an initial attempt at defining lung
injury in animals. We have identified four major features of
experimental ALI as well as measurements for each of those
features. We hope that this work will be of help to investigators and will also become a first step toward a definition of ALI
in animals.
This official summary of an ATS Workshop Proceedings was
prepared by an ad hoc subcommittee of the Assembly on
Microbiology, Tuberculosis, and Pulmonary Infections.
Writing Committee:
GUSTAVO MATUTE-BELLO, M.D. (Chair)
GREGORY P. DOWNEY, M.D., F.R.C.P(C). (Chair)
BETHANY B. MOORE, PH.D.
STEVE D. GROSHONG, M.D., PH.D
MICHAEL A. MATTHAY, M.D.
ARTHUR S. SLUTSKY, M.D.
WOLFGANG M. KUEBLER, M.D.
Author Disclosure: G.M-B. reported research support from the American Heart
Association ($50,001$100,000). G.P.D. reported no commercial interests or
non-governmental, non-commercial interests relevant to subject matter. B.B.M.
consulted with Centocor (up to $1,000) and received research support from the
American Lung Association ($100,000 or more), the Pulmonary Fibrosis Foundation ($50,001$100,000), and Centocor ($100,001 or more). S.D.G. and
M.A.M. each reported no commercial interests or non-governmental, noncommercial interests relevant to subject matter. A.S.S. served on advisory committees of Apeiron ($5,001$10,000) and Maquet Medical ($10,001$50,000) and
received royalties from Maquet Medical ($10,001$50,000). W.M.K. served on
advisory committees of Revotar ($1,001$5,000) and Hoffman-LaRoche
($1,001$5,000), received lecture fees from Nycomed ($1,001$5,000), and
received research support from GlaxoSmithKline ($10,001$50,000), HoffmanLaroche ($50,001$100,000) and Pfizer ($50,001$100,000). Note: the above
reflects disclosure by workshop report authors during period of document
development. Governmental support is not reported consistent with changes
in American Thoracic Society disclosure requirements effective September 2010.
Other workshop contributors also disclosed to ATS, in compliance with ATS
policies; summaries are available upon request.

The members of the subcommittee were:

GUSTAVO MATUTE-BELLO, M.D. (Chair)
GREGORY P. DOWNEY, M.D., F.R.C.P(C). (Chair)
EDWIN R. CHILVERS, PH.D., FMEDSCI.
CLAIRE DOERSCHUCK, M.D.
DIDIER DREYFUSS, M.D.
PETER Q. EICHACKER M.D.
JACK A. ELIAS, M.D.
ANDRES ESTEBAN, M.D.
NIALL D. FERGUSON, M.D., F.R.C.P.C.
ROBB W. GLENNY, M.D.
STEVE D. GROSHONG, M.D., PH.D.
PETER HENSON, PH.D., M.D.
WOLFGANG M. KUEBLER, M.D.
JOSE A. LORENTE, M.D.
THOMAS R. MARTIN, M.D.
MICHAEL A. MATTHAY, M.D.
BETHANY B. MOORE, PH.D.
JERRY A. NICK, M.D.

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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 44 2011

PETER RIMENSBERGER, M.D.

ARTHUR S. SLUTSKY, M.D.
STEFAN UHLIG, M.D.
TOM VAN DER POLL, M.D.
JESUS VILLAR, M.D., PH.D.
Acknowledgments: The authors thank Pam Henderson for her administrative
assistance and Mark Looney and Guy Zimmerman for reviewing a draft of the
manuscript.

References
1. Bernard GA, Artigas A, Brigham KL, Carlet J, Falke K, Hudson L,
Lamy M, LeGall JR, Morris A, Spragg R. Report of the AmericanEuropean consensus conference on acute respiratory distress syndrome: Definitions, mechanisms, relevant outcomes, and clinical
trial coordination. Consensus Committee. J Crit Care 1994;9:7281.
2. Katzenstein AL, Bloor CM, Leibow AA. Diffuse alveolar damagethe
role of oxygen, shock, and related factors. A review. Am J Pathol
1976;85:209228.
3. Irvin CG, Bates JH. Measuring the lung function in the mouse: the
challenge of size. Respir Res 2003;4:4.
4. Davies B, Morris T. Physiological parameters in laboratory animals and
humans. Pharm Res 1993;10:10931095.
5. Hamelmann E, Schwarze J, Takeda K, Oshiba A, Larsen GL, Irvin
CG, Gelfand EW. Noninvasive measurement of airway responsiveness in allergic mice using barometric plethysmography. Am J Respir
Crit Care Med 1997;156:766775.
6. Mestas J, Hughes CC. Of mice and not men: differences between
mouse and human immunology. J Immunol 2004;172:27312738.
7. Matute-Bello G, Frevert CW, Martin TR. Animal models of acute lung
injury. Am J Physiol Lung Cell Mol Physiol 2008;295:L379L399.
8. Swartz HM, Clarkson RB. The measurement of oxygen in vivo using
EPR techniques. Phys Med Biol 1998;43:19571975.
9. Bambot SB, Rao G, Romauld M, Carter GM, Sipior J, Terpetchnig E,
Lakowicz JR. Sensing oxygen through skin using a red diode laser
and fluorescence lifetimes. Biosens Bioelectron 1995;10:643652.
10. Martin EL, Sheikh TA, Leco KJ, Lewis JF, Veldhuizen RA. Contribution of alveolar macrophages to the response of the timp-3 null
lung during a septic insult. Am J Physiol Lung Cell Mol Physiol
2007;293:L779L789.
11. Bachofen M, Weibel ER. Structural alterations of lung parenchyma in
the adult respiratory distress syndrome. Clin Chest Med 1982;3:3556.
12. Bachofen A, Weibel ER. Alterations of the gas exchange apparatus in
adult respiratory insufficiency associated with septicemia. Am Rev
Respir Dis 1977;116:589615.
13. Villar J, Edelson JD, Post M, Mullen JB, Slutsky AS. Induction of heat
stress proteins is associated with decreased mortality in an animal
model of acute lung injury. Am Rev Respir Dis 1993;147:177181.
14. Herrera MT, Toledo C, Valladares F, Muros M, Diaz-Flores L, Flores
C, Villar J. Positive end-expiratory pressure modulates local and
systemic inflammatory responses in a sepsis-induced lung injury
model. Intensive Care Med 2003;29:13451353.
15. Sterner-Kock A, Vesely KR, Stovall MY, Schelegle ES, Green JF,
Hyde DM. Neonatal capsaicin treatment increases the severity of
ozone-induced lung injury. Am J Respir Crit Care Med 1996;153:436
443.
16. Matute-Bello G, Winn RK, Jonas M, Chi EY, Martin TR, Liles WC. Fas
(CD95) induces alveolar epithelial cell apoptosis in vivo: implications
for acute pulmonary inflammation. Am J Pathol 2001;158:153161.
17. Looney MR, Su X, Van Ziffle JA, Lowell CA, Matthay MA.
Neutrophils and their fc gamma receptors are essential in a mouse
model of transfusion-related acute lung injury. J Clin Invest 2006;
116:16151623.
18. Martin TR, Pistorese BP, Chi EY, Goodman RB, Matthay MA. Effects
of leukotriene b4 in the human lung. Recruitment of neutrophils into
the alveolar spaces without a change in protein permeability. J Clin
Invest 1989;84:16091619.
19. Wiener-Kronish JP, Albertine KH, Matthay MA. Differential responses of the endothelial and epithelial barriers of the lung in
sheep to escherichia coli endotoxin. J Clin Invest 1991;88:864875.
20. Berthiaume Y, Staub NC, Matthay MA. Beta-adrenergic agonists
increase lung liquid clearance in anesthetized sheep. J Clin Invest
1987;79:335343.
21. Erdmann AJ III, Vaughan TR Jr, Brigham KL, Woolverton WC, Staub
NC. Effect of increased vascular pressure on lung fluid balance in
unanesthetized sheep. Circ Res 1975;37:271284.

22. Vreim CR, Snashall PD, Demling RH, Staub NC. Lung lymph and free
interstitial fluid protein composition in sheep with edema. Am J
Physiol 1976;230:16501653.
23. Vreim CE, Snashall PD, Staub NC. Protein composition of lung fluids
in anesthetized dogs with acute cardiogenic edema. Am J Physiol
1976;231:14661469.
24. Brigham KL, Bowers R, Haynes J. Increased sheep lung vascular
permeability caused by escherichia coli endotoxin. Circ Res 1979;45:
292297.
25. Uhlig S, von Bethmann AN. Determination of vascular compliance,
interstitial compliance, and capillary filtration coefficient in rat isolated
perfused lungs. J Pharmacol Toxicol Methods 1997;37:119127.
26. Modelska K, Pittet JF, Folkesson HG, Courtney Broaddus V, Matthay
MA. Acid-induced lung injury. Protective effect of anti-interleukin-8
pretreatment on alveolar epithelial barrier function in rabbits. Am J
Respir Crit Care Med 1999;160:14501456.
27. Predescu D, Vogel SM, Malik AB. Functional and morphological
studies of protein transcytosis in continuous endothelia. Am J
Physiol Lung Cell Mol Physiol 2004;287:L895L901.
28. Sartori C, Matthay MA. Alveolar epithelial fluid transport in acute lung
injury: new insights. Eur Respir J 2002;20:12991313.
29. Matthay MA, Flori HR, Conner ER, Ware LB. Alveolar epithelial fluid
transport: basic mechanisms and clinical relevance. Proc Assoc Am
Physicians 1998;110:496505.
30. Tremblay L, Valenza F, Ribeiro SP, Li J, Slutsky AS. Injurious
ventilatory strategies increase cytokines and c-fos m-RNA expression in an isolated rat lung model. J Clin Invest 1997;99:944952.
31. Sakuma T, Pittet JF, Jayr C, Matthay MA. Alveolar liquid and protein
clearance in the absence of blood flow or ventilation in sheep. J Appl
Physiol 1993;74:176185.
32. Matthay MA, Folkesson HG, Clerici C. Lung epithelial fluid transport
and the resolution of pulmonary edema. Physiol Rev 2002;82:569600.
33. Hirsh M, Carmel J, Kaplan V, Livne E, Krausz MM. Activity of lung
neutrophils and matrix metalloproteinases in cyclophosphamidetreated mice with experimental sepsis. Int J Exp Pathol 2004;85:
147157.
34. Reutershan J, Morris MA, Burcin TL, Smith DF, Chang D, Saprito MS,
Ley K. Critical role of endothelial CXCR2 in LPS-induced neutrophil migration into the lung. J Clin Invest 2006;116:695702. Epub
2006 Feb 2016.
35. Nauseef WM, Olsson I, Arnljots K. Biosynthesis and processing of
myeloperoxidasea marker for myeloid cell differentiation. Eur J
Haematol 1988;40:97110.
36. Neff TA, Guo R-F, Neff SB, Sarma JV, Speyer CL, Gao H, Bernacki
KD, Huber-Lang M, McGuire S, Hoesel LM, et al. Relationship of
acute lung inflammatory injury to Fas/FasL system. Am J Pathol
2005;166:685694.
37. Lomas JL, Chung CS, Grutkoski PS, LeBlanc BW, Lavigne L, Reichner J,
Gregory SH, Doughty LA, Cioffi WG, Ayala A. Differential effects
of macrophage inflammatory chemokine-2 and keratinocyte-derived
chemokine on hemorrhage-induced neutrophil priming for lung
inflammation: assessment by adoptive cells transfer in mice. Shock
2003;19:358365.
38. Greenberger MJ, Strieter RM, Kunkel SL, Danforth JM, Goodman RE,
Standiford TJ. Neutralization of IL-10 increases survival in a murine
model of Klebsiella pneumonia. J Immunol 1995;155:722729.
39. Togbe D, Schnyder-Candrian S, Schnyder B, Doz E, Noulin N, Janot L,
Secher T, Gasse P, Lima C, Coelho FR, et al. Toll-like receptor and
tumour necrosis factor dependent endotoxin-induced acute lung
injury. Int J Exp Pathol 2007;88:387391.
40. Mukhopadhyay S, Hoidal JR, Mukherjee TK. Role of TNFalpha in
pulmonary pathophysiology. Respir Res 2006;7:125.
41. Ward PA. Role of complement, chemokines, and regulatory cytokines
in acute lung injury. Ann N Y Acad Sci 1996;796:104112.
42. Baugh JA, Bucala R. Macrophage migration inhibitory factor. Crit
Care Med 2002;30:S27S35.
43. Bulger EM, Maier RV. Lipid mediators in the pathophysiology of
critical illness. Crit Care Med 2000;28:N27-36.
44. Park WY, Goodman RB, Steinberg KP, Ruzinski JT, Radella F II, Park
DR, Pugin J, Skerrett SJ, Hudson LD, Martin TR. Cytokine balance
in the lungs of patients with acute respiratory distress syndrome. Am
J Respir Crit Care Med 2001;164:18961903.
45. Kobayashi Y. The role of chemokines in neutrophil biology. Front
Biosci 2008;13:24002407.
46. Wilborn J, Bailie M, Coffey M, Burdick M, Strieter R, Peters-Golden
M. Constitutive activation of 5-lipoxygenase in the lungs of

American Thoracic Society Documents

47.

48.
49.

50.

51.

52.

53.

54.
55.
56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.
68.

patients with idiopathic pulmonary fibrosis. J Clin Invest 1996;97:

18271836.
Feezor RJ, Baker HV, Mindrinos M, Hayden D, Tannahill CL,
Brownstein BH, Fay A, MacMillan S, Laramie J, Xiao W, et al.
Whole blood and leukocyte RNA isolation for gene expression
analyses. Physiol Genomics 2004;19:247254.
Fleige S, Pfaffl MW. Rna integrity and the effect on the real-time qRTPCR performance. Mol Aspects Med 2006;27:126139.
Glare EM, Divjak M, Bailey MJ, Walters EH. Beta-actin and GAPDH
housekeeping gene expression in asthmatic airways is variable and
not suitable for normalising mRNA levels. Thorax 2002;57:765770.
Bas A, Forsberg G, Hammarstrom S, Hammarstrom ML. Utility of the
housekeeping genes 18s rRNA, beta-actin and glyceraldehyde3-phosphate-dehydrogenase for normalization in real-time quantitative reverse transcriptase-polymerase chain reaction analysis of
gene expression in human T lymphocytes. Scand J Immunol 2004;
59:566573.
Schmittgen TD, Zakrajsek BA. Effect of experimental treatment on
housekeeping gene expression: validation by real-time, quantitative
RT-PCR. J Biochem Biophys Methods 2000;46:6981.
Bastarache JA, Ware LB, Bernard GR. The role of the coagulation
cascade in the continuum of sepsis and acute lung injury and acute
respiratory distress syndrome. Semin Respir Crit Care Med 2006;27:
365376.
Reutershan J, Ley K. Bench-to-bedside review: acute respiratory
distress syndromehow neutrophils migrate into the lung. Crit Care
2003;2004:453461.
Guo RF, Ward PA. Role of c5a in inflammatory responses. Annu Rev
Immunol 2005;23:821852.
Ohbayashi H. Matrix metalloproteinases in lung diseases. Curr Protein
Pept Sci 2002;3:409421.
Tsuno K, Miura K, Takeya M, Kolobow T, Morioka T. Histopathologic
pulmonary changes from mechanical ventilation at high peak airway
pressures. Am Rev Respir Dis 1991;143:11151120.
Frank JA, Pittet JF, Wray C, Matthay MA. Protection from experimental ventilator-induced acute lung injury by IL-1 receptor blockade. Thorax 2008;63:147153.
Sakashita A, Nishimura Y, Nishiuma T, Takenaka K, Kobayashi K,
Kotani Y, Yokoyama M. Neutrophil elastase inhibitor (sivelestat)
attenuates subsequent ventilator-induced lung injury in mice. Eur J
Pharmacol 2007;571:6271.
Hamanaka K, Jian MY, Weber DS, Alvarez DF, Townsley MI, AlMehdi AB, King JA, Liedtke W, Parker JC. Trpv4 initiates the acute
calcium-dependent permeability increase during ventilator-induced
lung injury in isolated mouse lungs. Am J Physiol Lung Cell Mol
Physiol 2007;293:L923L932.
Parker JC, Ivey CL, Tucker JA. Gadolinium prevents high airway
pressure-induced permeability increases in isolated rat lungs. J Appl
Physiol 1998;84:11131118.
de Prost N, Roux D, Dreyfuss D, Ricard JD, Le Guludec D, Saumon G.
Alveolar edema dispersion and alveolar protein permeability during
high volume ventilation: effect of positive end-expiratory pressure.
Intensive Care Med 2007;33:711717.
Peng X, Abdulnour RE, Sammani S, Ma SF, Han EJ, Hasan EJ, Tuder
R, Garcia JG, Hassoun PM. Inducible nitric oxide synthase contributes to ventilator-induced lung injury. Am J Respir Crit Care Med
2005;172:470479.
Eyal FG, Hamm CR, Parker JC. Reduction in alveolar macrophages
attenuates acute ventilator induced lung injury in rats. Intensive Care
Med 2007;33:12121218.
Yoshikawa S, Miyahara T, Reynolds SD, Stripp BR, Anghelescu M,
Eyal FG, Parker JC. Clara cell secretory protein and phospholipase
A2 activity modulate acute ventilator-induced lung injury in mice.
J Appl Physiol 2005;98:12641271.
von Bethmann AN, Brasch F, Nusing R, Vogt K, Volk HD, Muller KM,
Wendel A, Uhlig S. Hyperventilation induces release of cytokines from
perfused mouse lung. Am J Respir Crit Care Med 1998;157:263272.
Ricard J-D, Dreyfuss D, Saumon G. Production of inflammatory
cytokines in ventilator-induced lung injury: a reappraisal. Am J
Respir Crit Care Med 2001;163:11761180.
Dries DJ, Adams AB, Marini JJ. Time course of physiologic variables in
response to ventilator-induced lung injury. Respir Care 2007;52:3137.
Lutz C, Carney D, Finck C, Picone A, Gatto LA, Paskanik A,
Langenback E, Nieman G. Aerosolized surfactant improves pulmonary function in endotoxin-induced lung injury. Am J Respir Crit
Care Med 1998;158:840845.

737
69. Da J, Chen L, Hedenstierna G. Nitric oxide up-regulates the glucocorticoid receptor and blunts the inflammatory reaction in porcine
endotoxin sepsis. Crit Care Med 2007;35:2632.
70. Kemming GI, Flondor M, Hanser A, Pallivathukal S, Holtmannspotter
M, Kneisel FF, Reuter DA, Kisch-Wedel H, Zwissler B. Effects of
perfluorohexan vapor on gas exchange, respiratory mechanics, and
lung histology in pigs with lung injury after endotoxin infusion.
Anesthesiology 2005;103:585594.
71. Meyrick B, Brigham KL. Acute effects of escherichia coli endotoxin on
the pulmonary microcirculation of anesthetized sheep structure:
Function relationships. Lab Invest 1983;48:458470.
72. Rotta AT, Steinhorn DM. Partial liquid ventilation reduces pulmonary
neutrophil accumulation in an experimental model of systemic
endotoxemia and acute lung injury. Crit Care Med 1998;26:17071715.
73. Itoh T, Obata H, Murakami S, Hamada K, Kangawa K, Kimura H,
Nagaya N. Adrenomedullin ameliorates lipopolysaccharide-induced
acute lung injury in rats. Am J Physiol Lung Cell Mol Physiol 2007;
293:L446L452.
74. Goggel R, Winoto-Morbach S, Vielhaber G, Imai Y, Lindner K, Brade
L, Brade H, Ehlers S, Slutsky AS, Schutze S, et al. Paf-mediated
pulmonary edema: a new role for acid sphingomyelinase and
ceramide. Nat Med 2004;10:155160.
75. Huang YQ, Sauthoff H, Herscovici P, Pipiya T, Cheng J, Heitner S,
Szentirmai O, Carter B, Hay JG. Angiopoietin-1 increases survival
and reduces the development of lung edema induced by endotoxin
administration in a murine model of acute lung injury. Crit Care Med
2008;36:262267.
76. Kolosova IA, Mirzapoiazova T, Moreno-Vinasco L, Sammani S, Garcia
JG, Verin AD. Protective effect of purinergic agonist ATPgammas
against acute lung injury. Am J Physiol Lung Cell Mol Physiol 2008;
294:L319L324.
77. Tzeng HP, Ho FM, Chao KF, Kuo ML, Lin-Shiau SY, Liu SH. Betalapachone reduces endotoxin-induced macrophage activation and
lung edema and mortality. Am J Respir Crit Care Med 2003;168:
8591.
78. Takeoka M, Sakai A, Ueda G, Ge RL, Ishizaki T, Panos RJ, Taniguchi
S. Dibutyryl camp inhibits endotoxin-induced increases in pulmonary vascular resistance and fluid filtration coefficient in the perfused
rat lung. Tohoku J Exp Med 1997;183:273284.
79. Chu CH, David Liu D, Hsu YH, Lee KC, Chen HI. Propofol exerts
protective effects on the acute lung injury induced by endotoxin in
rats. Pulm Pharmacol Ther 2007;20:503512.
80. Urbain B, Gustin P, Ansay M. Endotoxin-induced microvascular injury
in isolated and perfused pig lungs. Vet Res Commun 1992;16:453464.
81. Mouchawar AM, Kavanagh BP, Pearl RG. Serum but not plasma
produces injury in the perfused rabbit lung. Anesth Analg 1994;79:
4045.
82. Winn R, Nickelson S, Rice CL. Fluid filtration coefficient of isolated
goat lungs was unchanged by endotoxin. J Appl Physiol 1988;64:
24632467.
83. Chlopicki S, Walski M, Bartus JB. Ultrastructure of immediate
microvascular lung injury induced by bacterial endotoxin in the
isolated, no-deficient lung perfused with full blood. J Physiol
Pharmacol 2005;56:4764.
84. Kuebler WM, Borges J, Sckell A, Kuhnle GE, Bergh K, Messmer K,
Goetz AE. Role of l-selectin in leukocyte sequestration in lung
capillaries in a rabbit model of endotoxemia. Am J Respir Crit Care
Med 2000;161:3643.
85. Lutz CJ, Picone A, Gatto LA, Paskanik A, Landas S, Nieman GF.
Exogenous surfactant and positive end-expiratory pressure in the
treatment of endotoxin-induced lung injury. Crit Care Med 1998;26:
13791389.
86. Mizgerd JP, Skerrett SJ. Animal models of human pneumonia. Am J
Physiol Lung Cell Mol Physiol 2008;294:L387L398.
87. Bryson DG, Ball HJ, McAliskey M, McConnell W, McCullough SJ.
Pathological, immunocytochemical and microbiological findings in
calf pneumonias associated with haemophilus somnus infection.
J Comp Pathol 1990;103:433445.
88. Choi G, Hofstra JJ, Roelofs JJ, Rijneveld AW, Bresser P, van der Zee
JS, Florquin S, van der Poll T, Levi M, Schultz MJ. Antithrombin
inhibits bronchoalveolar activation of coagulation and limits lung
injury during Streptococcus pneumoniae pneumonia in rats. Crit
Care Med 2008;36:204210.
89. Kondo H, Taguchi M, Abe N, Nogami Y, Yoshioka H, Ito M.
Pathological changes in epidemic porcine Pneumocystis carinii
pneumonia. J Comp Pathol 1993;108:261268.

738

AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 44 2011

90. Mizgerd JP, Scott ML, Spieker MR, Doerschuk CM. Functions of Ikb
proteins in inflammatory responses to Escherichia coli LPS in mouse
lungs. Am J Respir Cell Mol Biol 2002;27:575582.
91. Mizgerd JP, Spieker MR, Doerschuk CM. Early response cytokines
and innate immunity: essential roles for TNF receptor 1 and type I
IL-1 receptor during Escherichia coli pneumonia in mice. J Immunol
2001;166:40424048.
92. Ong ES, Gao XP, Xu N, Predescu D, Rahman A, Broman MT, Jho DH,
Malik ABE. Coli pneumonia induces CD18-independent airway
neutrophil migration in the absence of increased lung vascular permeability. Am J Physiol Lung Cell Mol Physiol 2003;285:L879L888.
93. Viget NB, Guery BP, Ader F, Neviere R, Alfandari S, Creuzy C,
Roussel-Delvallez M, Foucher C, Mason CM, Beaucaire G, et al.
Keratinocyte growth factor protects against Pseudomonas aeruginosainduced lung injury. Am J Physiol Lung Cell Mol Physiol 2000;279:
L1199L1209.
94. Wang Q, Teder P, Judd NP, Noble PW, Doerschuk CM. CD44 deficiency
leads to enhanced neutrophil migration and lung injury in Escherichia
coli pneumonia in mice. Am J Pathol 2002;161:22192228.
95. Mizgerd JP, Lupa MM, Hjoberg J, Vallone JC, Warren HB, Butler JP,
Silverman ES. Roles for early response cytokines during Escherichia
coli pneumonia revealed by mice with combined deficiencies of all
signaling receptors for TNF and IL-1. Am J Physiol Lung Cell Mol
Physiol 2004;286:L1302L1310.

96. Teder P, Vandivier RW, Jiang D, Liang J, Cohn L, Pure E, Henson

PM, Noble PW. Resolution of lung inflammation by CD44. Science
2002;296:155158.
97. Sawa T, Corry DB, Gropper MA, Ohara M, Kurahashi K, WienerKronish JP. IL-10 improves lung injury and survival in Pseudomonas
aeruginosa pneumonia. J Immunol 1997;159:28582866.
98. Seeger W, Obernitz R, Thomas M, Walmrath D, Suttorn N, Holland
IB, Grimminger F, Eberspacher B, Hugo F, Bhakdi S. Lung
vascular injury after administration of viable hemolysin-forming
Escherichia coli in isolated rabbit lungs. Am Rev Respir Dis 1991;
143:797805.
99. Song GW, Robertson B, Curstedt T, Gan XZ, Huang WX. Surfactant
treatment in experimental Escherichia coli pneumonia. Acta Anaesthesiol Scand 1996;40:11541160.
100. Su X, Robriquet L, Folkesson HG, Matthay MA. Protective effect of
endogenous beta-adrenergic tone on lung fluid balance in acute
bacterial pneumonia in mice. Am J Physiol Lung Cell Mol Physiol
2006;290:L769L776.
101. Luna CM, Baquero S, Gando S, Patron JR, Morato JG, Sibila O,
Absi R, Famiglietti A, Vay CA, Von Stecher F, et al. Experimental severe Pseudomonas aeruginosa pneumonia and antibiotic
therapy in piglets receiving mechanical ventilation. Chest 2007;
132:523531.